Field of Invention
The present invention relates to the field of double-stranded RNA (dsRNA)-mediated gene silencing in insect species. More particularly, the present invention relates to genetic constructs designed for the expression of dsRNA corresponding to novel target genes in the insect pest Lymantria dispar, commonly known as the gypsy moth. These constructs are particularly useful in RNAi-mediated control of the gypsy moth.
Background
Lymantria dispar (Lepidoptera: Erebidae), the gypsy moth, is the most serious insect pest of the North American forests where larvae feed on over 300 tree and shrub species, especially hardwood and shade trees. Measures to control the pest and reduce its spread have been implemented in the United States, including augmentative release of natural enemies, use of chemical pesticides (acephate=Orthene®, carbaryl=Sevin®, and diflubenzuron=Dimilin®), mating disruption using a chemical pheromone (Disparlure), and use of microbial biopesticides such as the nucleopolyhedrovirus (Gypchek®) or the gram-positive soil bacterium Bacillus thuringiensis (Bt) (Höfte & Whiteley, Microbiol. Revs., (1989) 53:2, 242-55). These measures have been employed with variable success and resistance to biopesticides has evolved rapidly in larval populations. Recently publicly available data sets for gypsy moth-associated genes were generated through characterization of transcriptomes of both an L. dispar-derived cell line IPLB-Ld652Y (Sparks & Gundersen-Rindal, Viruses, (2011) 3:12, 2339-50) and the L. dispar larval midgut (Sparks et al., PloS One, (2013) 8:5, e61190).
The discovery of RNA mediated interference (RNAi) has facilitated research to understand gene function and regulation. RNAi provides a silencing technique to deplete functional expression of targeted genes by degradation of mRNA into short RNAs. RNAi takes advantage of internal cellular defenses against the presence of dsRNA, which typically indicates an on-going viral infection. Double stranded RNAs (dsRNA) are cleaved by Dicer, a member of the RNase III superfamily of bidentate nucleases that are evolutionarily conserved in worms, flies, plants, fungi and mammals (Bernstein et al., Nature (2001) 409:6818, 363-66; Ketting et al., Genes Develop, (2001) 15:20, 2654-59). These 19-21 base pair short RNAs or siRNAs, unwind and together with RNA-induced silencing complex (RISC) associate with the complementary RNA. This RISC-RNA complex in conjunction with argonaute multi-domain protein containing an RNAse H like domain is responsible for target degradation and silencing the gene (Martinez et al., Cell, (2002) 110:5, 563-74; Bartel, Cell, (2004) 116:2, 281-97).
The major limiting factors for in vivo application of dsRNA mediated RNAi have been the cellular uptake of dsRNA, the method of delivery, and the stability of dsRNA after its delivery. Effective approaches for RNAi delivery—such as injecting the target organism, soaking the target, or feeding extracellular dsRNA (or intracellular dsRNA in a transgenic microbe) to the target—have been reported with varied success. Fire and colleagues first observed the phenomenon of RNAi by injecting dsRNA in the nematode, Caenorhabditis elegans (C. elegans) to deplete unc-22 gene generating twitching phenotypes (Fire et al., Nature, (1998) 391:6669, 806-11). Later, unc-22 depletion was accomplished in C. elegans by feeding E. coli inducing dsRNA (Timmons & Fire, Nature, (1998) 395:6705, 854). Subsequently RNAi was successfully performed in other animals such as Drosophila melanogaster (D. melanogaster) to downregulate the frizzled and frizzled 2 genes (Kennerdell & Carthew, Cell, (1998) 95:7, 1017-26). Clemens et al, used RNAi in Drosophila S2 tissue cultures cells to inhibit the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) which prevented the activation of the downstream ERK-A (MAPK) in the insulin signal transduction pathway and vice versa (Clemens et al., Proc Nat'l Acad Sci USA, (2000) 97:12, 6499-6503).
Though lepidopteran insects have been resistant to RNAi, some successful dsRNA mediated RNAi instances have been observed in this class of insects. In one instance, aminopeptidase gene (slapn) dsRNA injected into the midgut of Spodoptera litura reduced its expression and susceptibility to Bt cry1C protein (Rajagopal et al., J Biol Chem, (2002) 277:49, 46849-51). Other studies demonstrated RNAi by feeding dsRNA either in a sucrose droplet to reduce the transcription of carboxylesterase gene (EposCXE1) in Epiphyas postvittana, or from plants engineered to produce dsRNA against cytochrome P450 gene (CYP6AE14) in Helicoverpa armigera (Turner et al., Insect Mol Biol, (2006) 15:3, 383-91; Mao et al., Nature Biotech, (2007) 25:11, 1307-13).
Disclosed herein, we describe various specifically designed RNAi examined in vivo in L. dispar larvae for efficiency in gene knock-down and depletion; several different delivery methods were also evaluated. Observations revealed that dsRNA synthesized to the midgut genes of L. dispar and delivered per os could suppress the expression of specifically targeted genes in vivo. RNA interference through feeding may be beneficial over other techniques, firstly due to the ease in feeding large number of insects inexpensively and secondly to preserve the sustainable farming practices.